Method for assisting determination of depression

Abstract

[Problem] To provide: a method for identifying a new depression marker and assisting determination of depression on the basis of the depression marker; and diagnosis kit for depression based on the depression marker. [Solution] This method for assisting the determination of depression is characterized by including a step for measuring the amount of von Willebrand factor present in extracellular vesicles in a biological sample collected from a subject and comparing the measured amount with a predetermined value.

Description

Methods to help identify depression

[0001] The present invention relates to a method for assisting in the diagnosis of depression, a kit for diagnosing depression, and use for diagnosing depression.

[0002] The total number of patients with psychiatric disorders in Japan exceeds 4 million, of which mood disorders, including depression, have the largest number of patients and have become a major social problem. While investigative methods for diagnosing depression use indicators such as DSM-5-TR and ICD-11, the subjective judgment element of the physician cannot be eliminated, and evaluation and diagnostic methods based on objective measurement data are needed. Optical topography, which numerically diagnoses depression based on changes in cerebral blood flow, is also used as an objective testing method, but its use is limited to assisting in the differentiation of depression, bipolar disorder, and schizophrenia.

[0003] Objective, quantitative biomarkers that reflect changes in the patient's body are a powerful tool for understanding and diagnosing the pathophysiology of depression. To date, various methods have been disclosed, including a method for diagnosing depression using the methylation level of a specific DNA sampled from blood as an index (see Patent Document 1), a method for diagnosing depression by comparing the expression level of a specific gene with a reference value (see Patent Document 2), and a method for assessing the severity of depression using specific metabolites such as 4-aminobutyric acid (γ(gamma)-aminobutyric acid: GABA), arginine, and argininosuccinic acid as biomarkers (see Patent Document 3).

[0004] Exosomes are small vesicles of approximately 100 nm secreted by cells. They contain information about the cells that secreted them, such as DNA, proteins, and microRNAs. Therefore, they are expected to be used as biomarkers for various diseases. For example, a method for assisting the diagnosis of depression, which involves measuring the level of specific microRNAs contained in exosomes, has been disclosed (see Patent Document 4). However, none of these biomarkers have yet been put to clinical use. Furthermore, it was unclear whether remission could be objectively measured and evaluated.

[0005] Japanese Patent Application Laid-Open No. 2019-193578 Japanese Patent Application Laid-Open No. 2017-000063 International Publication No. 2017 / 082103 Pamphlet Japanese Patent Application Laid-Open No. 2021-112167

[0006] An object of the present invention is to find a new depression marker, provide a method for assisting in the diagnosis of depression based on the depression marker, and provide a diagnostic kit for depression based on the depression marker.

[0007] The present inventors have been diligently searching for biomarkers for depression to solve the above-mentioned problems, and have focused on glycoproteins on exosomes collected from peripheral blood. They have discovered that measuring or evaluating von Willebrand factor (hereinafter also referred to as "vWF"), a glycoprotein on exosomes, makes it possible to determine the state of depression and the state of remission due to treatment, and have completed the present invention.

[0008] That is, the present invention is as follows: [1] A method for assisting in the diagnosis of depression, comprising the step of measuring the amount of von Willebrand factor present in extracellular vesicles in a biological sample collected from a subject and comparing the amount with a predetermined value. [2] The method for assisting in the diagnosis of depression according to [1] above, wherein the von Willebrand factor is glycosylated von Willebrand factor. [3] The method for assisting in the diagnosis of depression according to [1] or [2] above, wherein the amount of glycosylated von Willebrand factor is measured using a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the glycan. [4] The method for assisting in the diagnosis of depression according to [3] above, wherein the lectin is wheat germ agglutinin (WGA). [5] The method for assisting in the diagnosis of depression according to any of [1] to [4] above, wherein the biological sample is plasma, serum, or blood. [6] The method for assisting in the diagnosis of depression according to any one of [1] to [5] above, characterized in that a subject suffering from depression or with a history of being diagnosed with depression is diagnosed as to whether or not the depression has gone into remission. [7] A kit for discriminating depression, comprising an anti-von Willebrand factor antibody or a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the sugar chain of glycosylated von Willebrand factor. [8] The kit for discriminating depression according to [7] above, comprising an anti-von Willebrand factor antibody and a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the sugar chain of glycosylated von Willebrand factor. [9] The kit for discriminating depression according to [7] or [8] above, characterized in that the lectin is wheat germ agglutinin (WGA).

[10] The kit for discriminating depression according to any one of [7] to [9] above, characterized in that the anti-von Willebrand factor antibody is an anti-von Willebrand factor antibody not bound to a sugar chain.

[11] Use of an anti-von Willebrand factor antibody or a lectin that recognizes the sugar chain of von Willebrand factor for diagnosing depression.

[0009] The present invention makes it possible to objectively determine depression. Furthermore, in the follow-up observation of a patient with depression, it becomes possible to determine whether or not the patient has entered a remission period.

[0010] Figure 1 shows the results of scanning electron microscopy observation of blood exosomes derived from healthy subjects (HS), patients with depression (acute phase: DP), and patients with depression (remission phase: REM) in Example 1. Figure 2 shows the average particle size (nm), protein concentration (μg / mL), and 10 ９The figure shows the results of examining the amount of protein (μg) per particle. Figure 3 shows the results of examining the protein sugar chain patterns on exosomes derived from five healthy subjects and five patients with acute depression by lectin blotting in Example 2. Figure 4 shows the results of examining the signal intensity of bands in healthy subjects and patients with acute depression, focusing on bands around 75 kDa or 250 kDa in lectin blotting in Example 2. The left figure in Figure 4 shows the signal intensity of the band around 75 kDa, and the right figure in Figure 4 shows the signal intensity of the band around 250 kDa. Figure 5 shows the results of lectin blot analysis of patients with acute depression and patients with remission of depression in Example 3. The left figure in Figure 6 shows the results of measuring the signal intensity of the band around 250 kDa and comparing the same patients with acute depression and patients with remission of depression in Example 3. The right panel of Figure 6 shows the results of receiver operating characteristic (ROC) analysis based on the signal intensities of bands from the same patient with acute depression and patient with remission of depression in Example 3. The left panel of Figure 7 shows the results of measuring and comparing the signal intensities of bands around 250 kDa in healthy subjects, patients with acute depression, and patients with remission of depression based on the demographic data in Table 1 in Example 3. The right panel of Figure 7 shows the results of ROC analysis based on the signal intensities of bands from healthy subjects and patients with acute depression in Example 3. The left panel of Figure 8 shows the results of measuring and comparing the signal intensities of bands around 250 kDa in healthy subjects and patients with depression based on the demographic data in Table 2 in Example 3. The right panel of Figure 8 shows the results of ROC analysis based on the signal intensities of bands from healthy subjects and patients with depression in Example 3. Figure 9 shows a photograph of a stained gel after separation by SDS-PAGE in Example 4. Figure 10 shows the results of ELISA analysis of von Willebrand factor (vWF) on exosomes derived from healthy subjects, patients with depression (acute phase), and patients with depression (remission phase) based on the demographic data in Table 1 in Example 4. Figure 11 shows the results of ELISA analysis of vWF on exosomes derived from healthy subjects and patients with depression (acute phase) based on the demographic data in Table 2 in Example 4.Figure 12 shows the results of analyzing glycosylated vWF protein on exosomes derived from healthy subjects, patients with acute depression, and patients with remission of depression by sandwich ELISA using lectin WGA based on the demographic data in Table 1 in Example 5. Figure 13 shows the results of ROC analysis of healthy subjects, patients with acute depression, and the same patient with depression in the remission and acute phases in Example 5. Figure 14 shows the results of analyzing glycosylated vWF protein on exosomes derived from healthy subjects and patients with acute depression by sandwich ELISA using lectin WGA based on the demographic data in Table 2 in Example 5. Figure 15 shows the results of analyzing glycosylated vWF protein on exosomes derived from healthy subjects, patients with acute depression, and patients with remission of depression based on the demographic data in Table 1, and patients with bipolar disorder based on the demographic data in Table 2 in Table 4, based on the demographic data in Table 2 in Example 6, using lectin WGA by sandwich ELISA. Figure 16 shows the results of ELISA analysis of vWF protein in plasma obtained from 20 healthy subjects (HS) and 21 patients with acute depression (MDD-DP) based on the demographic data in Table 2.

[0011] The method for assisting in the diagnosis of depression of the present invention is characterized by a step of measuring the amount of von Willebrand factor present in extracellular vesicles in a biological sample collected from a subject and comparing the amount with a predetermined value, and is hereinafter also referred to as the "method for assisting in the diagnosis of depression." The method for assisting in the diagnosis of depression does not include a step of diagnosis by a physician and is not a method for diagnosing humans. Furthermore, the kit for diagnosing depression of the present invention is not particularly limited as long as it contains an anti-von Willebrand factor antibody or a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the glycan of glycan-bound von Willebrand factor, and is hereinafter also referred to as the "kit for diagnosing depression." Furthermore, the use of the present invention for diagnosing depression is not particularly limited as long as it uses an anti-von Willebrand factor antibody or a lectin that recognizes the glycan of von Willebrand factor for diagnosing depression, and is hereinafter also referred to as the "use for diagnosing depression."

[0012] In this specification, biological samples include blood and blood-related samples derived therefrom (blood, serum, plasma, etc.), lymph, sweat, tears, saliva, urine, and feces, with blood-related samples being preferred. Furthermore, subjects include mammals, such as humans, dogs, cats, monkeys, rats, mice, and rabbits.

[0013] (von Willebrand Factor) In this specification, von Willebrand factor (vWF) is a blood coagulation factor known as a factor, and its basic unit has a molecular weight of approximately 250,000. This vWF forms dimers with a molecular weight of approximately 500,000, which polymerize to form multimers, which are involved in hemostatic function.

[0014] The vWF present in the extracellular vesicles may be vWF bound to the surface of the extracellular vesicles, vWF encapsulated in the extracellular vesicles, or vWF penetrating the membrane of the extracellular vesicles. Examples of the extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles.

[0015] The extracellular vesicles can be separated from biological samples by known methods such as centrifugation, ultracentrifugation, and filtration, or can be separated using a commercially available extracellular vesicle separation kit. Alternatively, they can be obtained by concentrating membrane vesicles based on extracellular vesicle markers such as CD9, CD63, CD41a, CD41b, CD42b, CD61, CD62P, and CD81.

[0016] The vWF may be a glycosylated vWF, and the glycosylated vWF may be an N-glycan in which a glycosylated vWF is attached to an asparagine side chain, or an O-glycan in which a glycosylated vWF is attached to a serine or threonine side chain, but is preferably an N-glycan. In the case of an N-glycan, it may be a complex glycosylated vWF, a hybrid glycosylated vWF, or a high-mannose glycosylated vWF, but is preferably a complex glycosylated vWF or a hybrid glycosylated vWF. Furthermore, the glycosylated vWF may have N-acetylglucosamine or sialic acid in the branched chain of the glycosylated vWF, preferably N-acetylglucosamine and sialic acid.

[0017] (Diagnosing depression) In this specification, diagnosing depression means determining whether a subject suspected of having depression is in a depressive state, or determining whether a subject suffering from depression or who has a history of being diagnosed with depression has recovered from depression. In this specification, subjects suspected of having depression may include bipolar disorder patients or patients suspected of having bipolar disorder.

[0018] (Measurement of the amount of vWF) In the present specification, the method for measuring the amount of vWF is not particularly limited, and examples thereof include ELISA (Enzyme-Linked Immunosorbent Assay), sandwich ELISA, enzyme-linked immunosorbent assay (EIA), latex agglutination, Western blotting, immunochromatography, CBA (Cytometric Bead Assay), immunoprecipitation, RIA, and gold colloid assay using an antibody that binds to vWF (anti-vWF antibody), a lectin that recognizes the sugar chain of vWF, or an aptamer that binds to the sugar chain of vWF. Furthermore, the antibody may be immobilized on a microplate, a plastic tube, glass beads, or the like. Furthermore, when a lectin is used, examples thereof include lectin blotting.

[0019] The anti-vWF antibody is preferably an antibody that specifically binds to the vWF, and an antibody that specifically binds to the vWF means that it binds to the protein portion (core protein portion) of vWF and / or the glycan portion of vWF, but does not bind or does not substantially bind to the protein portion (core protein portion) of proteins other than vWF and / or the glycan portion of proteins other than vWF.

[0020] The anti-vWF antibody can be produced by techniques well known to those skilled in the art, such as the hybridoma method or the phage display method. Alternatively, the vWF gene sequence may be obtained from a database such as the National Center for Biotechnology Information (NCBI), a recombinant vWF protein may be synthesized by cloning, and a mouse may be immunized with the synthesized recombinant vWF protein to produce the antibody. Furthermore, a commercially available antibody may also be used.

[0021] The anti-vWF antibody may be any type of antibody, including human antibodies, chimeric antibodies, humanized antibodies, and F(ab') ２ , Fab, diabody, Fv, ScFv, or Sc(Fv) ２ Examples of antibody fragments include those described above. The above antibodies may be polyclonal or monoclonal. Chimeric or humanized antibodies can be produced by genetic engineering in accordance with standard methods. Antibody fragments can also be produced by digesting full-length antibodies with pepsin or papain, for example.

[0022] The lectin that recognizes the vWF sugar chain is not particularly limited as long as it can recognize the vWF sugar chain. However, it is preferably a lectin that recognizes sugar chains bound to vWF protein, preferably N-acetylglucosamine (GlcNAc) or sialic acid (NeuNAc) in the branched chains of N-glycans bound to vWF protein, and more preferably a lectin that recognizes N-acetylglucosamine and sialic acid in N-glycans. A specific example is wheat germ agglutinin (WGA) lectin. This WGA is a lectin that recognizes N-acetylglucosamine (GlcNAc) and sialic acid in the branched chains of sugar chains, particularly N-acetylneuraminic acid among sialic acids. This WGA lectin may be wild-type WGA or a mutant WGA, but wild-type WGA is preferred. The lectin that recognizes the sugar chain refers to a lectin that forms hydrogen bonds with sugar chains and recognizes sugar chains through the involvement of hydrophobic interactions, van der Waals interactions, etc.

[0023] The anti-vWF antibody or the lectin that recognizes the sugar chain of vWF may be labeled with a labeling substance. Examples of such a labeling substance include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and red fluorescent protein (Red Fluorescent Protein). Examples of the fluorescent substance include fluorescent protein (RFP), fluorescent proteins such as luciferase, radioisotopes such as 3H, 14C, 125I or 131I, metal colloids, non-metal colloids, dye particles such as dye sols or dispersed dyes, latex particles or colored microparticles, biotin, avidin, or chemiluminescent substances.

[0024] (Discrimination of Depression) Depression (acute phase depression) can be determined by comparing the amount of vWF present in extracellular vesicles in a biological sample collected from a subject with a predetermined value. For example, the amounts of vWF present in extracellular vesicles in biological samples collected from healthy subjects and depressed patients (vWF amount in healthy subjects and vWF amount in depressed patients) are measured in advance to obtain predetermined values. Next, the amount of vWF present in extracellular vesicles in a biological sample collected from the subject (vWF amount in subjects) is measured, and the determination can be made by comparing whether the vWF amount in subjects is closer to the vWF amount in healthy subjects or the vWF amount in depressed patients. Alternatively, a threshold may be calculated based on the vWF amount in healthy subjects and the vWF amount in depressed patients as the predetermined value. If the vWF amount in a subject is less than the predetermined threshold, the subject can be determined to be in a depressed state, and if it is equal to or greater than the predetermined threshold, the subject can be determined not to be in a depressed state. Here, if the subject suffers from depression or has a history of being diagnosed with depression, the subject can be determined to be in a depressed state if the amount of vWF in the biological sample is below a predetermined threshold, and the subject can be determined to be not in a depressed state at the testing stage, i.e., in a remission state, if the amount is equal to or greater than the predetermined threshold. Examples of the extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles. The amount of vWF may be the absolute amount of vWF or the relative amount of vWF, for example, the concentration of vWF.

[0025] The method for determining the predetermined threshold is not particularly limited, and examples include a method in which the vWF amount in healthy individuals and the vWF amount in patients with depression are used as indicators. In this case, the predetermined threshold may be determined with reference to the average or median vWF protein level in biological samples from each patient. Specifically, when plasma is used as a biological sample and the vWF amount in the plasma is measured using an anti-vWF antibody, a method in which the vWF protein concentration in the plasma is set to 0.15 μg / 500 μL, 0.2 μg / 500 μL, or 0.24 μg / 500 μL as a threshold and compared with the vWF protein concentration in the plasma of the subject can be used for determination.

[0026] Another example of a method for determining the amount of glycosylated vWF present in extracellular vesicles in plasma using plasma as a biological sample and WGA lectin can be used. The concentration of glycosylated vWF protein present in extracellular vesicles in plasma can be compared with the concentration of glycosylated vWF protein present in extracellular vesicles in the subject's plasma using threshold values ​​of 50 μg / 500 μL, 100 μg / 500 μL, and 150 μg / 500 μL.

[0027] Alternatively, the predetermined threshold value may be determined from the vWF amount in healthy individuals and the vWF amount in patients with depression using a Youden index based on an ROC curve (Receiver Operating Characteristic curve). Furthermore, immunochromatography may be used to make it possible to determine the amount of vWF present in extracellular vesicles in a biological sample collected from a subject visually or under a fluorescence microscope using, for example, fluorescence from an anti-vWF antibody or a fluorescent substance labeled with a lectin that recognizes the sugar chain of vWF.

[0028] The present kit for diagnosing depression may contain an anti-von Willebrand factor antibody or a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chains of the sugar chains of glycosylated von Willebrand factor, and depression can be diagnosed by measuring the amount of von Willebrand factor present in extracellular vesicles in a biological sample collected from a subject using a vWF antibody or the above-mentioned lectin. The present kit for diagnosing depression preferably contains a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chains of glycosylated von Willebrand factor, preferably a lectin that recognizes N-acetylglucosamine and sialic acid in the branched chains of glycosylated von Willebrand factor, more preferably lectin WGA, and more preferably contains an anti-von Willebrand factor antibody and the above-mentioned lectin. The use of such a lectin enables more accurate diagnosis of depression. In addition, when the present depression discrimination kit contains an anti-von Willebrand factor antibody and a lectin, it is preferable to use an anti-von Willebrand factor antibody that does not have a sugar chain bound to it.

[0029] In addition, the present depression diagnosis kit may further include reagents for measuring the amount of vWF using the ELISA method or the like, a secondary antibody that acts on the anti-vWF antibody or lectin used to measure the amount of vWF, a substrate reagent that produces color or fluorescence, a reagent for separating extracellular vesicles from a biological sample, and an instruction manual describing a method for measuring the amount of vWF.

[0030] The present use for diagnosing depression is the use of an anti-von Willebrand factor antibody or a lectin that recognizes the glycan of vWF for diagnosing depression, and the anti-vWF antibody or the lectin that recognizes the glycan of vWF can be used for diagnosing depression. Furthermore, von Willebrand factor, particularly glycan-bound von Willebrand factor, can also be used as a biomarker to assist in diagnosing depression.

[0031] The contents of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

[0032] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0033] 1. Experimental Methods [Subjects] The Institutional Review Board of Yamaguchi University Hospital approved the following experiment, and all subjects provided written informed consent to participate. The following experiment was conducted in accordance with the latest version of the Declaration of Helsinki.

[0034] Patients with major depressive disorder (MDD) and healthy controls were recruited using the following method. MDD patients were referred from Yamaguchi University Hospital or nearby clinics and hospitals. All subjects were recruited between April 2012 and June 2013 and followed up until August 2014. MDD patients were selected and diagnosed using a structured clinical interview, including the Japanese version of the International Neuropsychiatric Interview (MINI) 5.0.0. Depression severity was assessed using the Hamilton Depression Rating Scale (HDRS). Social functioning was assessed using the Global Assessment of Functioning (GAF). The Mini-Mental State Examination (MMSE) was used to exclude patients with possible dementia (scores below 24). Patients with depression (in remission) (MDD-REM) were classified according to the criteria for complete remission in the DSM-5. Healthy subjects were recruited through local advertisements and selected using the MINI and clinical interviews. Healthy subjects with a family history of psychiatric illness were excluded from the study. The demographic data of the MDD patients and healthy subjects who participated in this study are summarized in Tables 1 and 2.

[0035]

[0036] The 10 patients with depression (in remission) (MDD-REM) in Table 1 are patients who achieved remission in the subsequent follow-up of the 11 patients with depression (in acute phase) (MDD-DP). Therefore, 10 of the 11 MDD-DP patients and the 10 MDD-REM patients are the same patients.

[0037] Table 2 shows the demographic data obtained by adding 10 more healthy subjects and 10 more patients with acute depression (MDD-DP) to the demographic data in Table 1, resulting in 20 healthy subjects (HC) and 21 patients with acute depression (MDD-DP).

[0038] Isolation of Extracellular Vesicles (EVs) from Human Plasma. Venous fasting blood was collected from subjects between 9:00 AM and 12:00 PM. Blood samples were centrifuged at 2400 × g for 5 minutes to separate plasma and peripheral blood cells. These samples were stored at −80°C until use. 500 μL of processed plasma was directly overlaid onto a qEV size-exclusion column (qEV Original-70 Gen2, Izon Science), after which the sample was concentrated to a final volume of 400 μL. Protein concentrations of each qEV fraction were estimated using the Micro BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's protocol. To identify the enriched EV fraction, Western blotting was performed using antibodies against CD9 and CD63 as EV markers, and it was confirmed that the enriched fraction was an EV fraction.

[0039] Nanoparticle Tracking Analysis (NTA): Particle size distribution and concentration were measured using Videodrop (Myriad). 7 μL of each EV fraction was used to measure particle concentration and size in each condition. To remove macroparticles, the threshold was set to 4.2, and the exposure time for each frame was 0.90 ms. Accumulation was performed to count a sufficient number of particles, so that 100-300 particles were counted in each condition.

[0040] Enzyme-linked immunosorbent assay (ELISA): Plasma EVs were mixed equally with M-PER (Thermo Scientific), a protein solubilizer, to separate proteins from plasma EVs for ELISA analysis. Samples were assayed using a VWF Human ELISA Kit (#EHVWF, Invitrogen) according to the manufacturer's instructions.

[0041] Sandwich ELISA: To construct a sandwich ELISA to quantify vWF recognized by WGA, a 96-well plate coated with an antibody against human vWF (included in the VWF Human ELISA Kit) was treated with PNGase F (P0704, New England Biolabs) for 4 hours at 37°C to remove the N-linked glycans of the antibody against human vWF, preventing WGA-biotin lectin from binding to the glycans of the anti-human vWF antibody. After washing, plasma EVs were added to the plate and incubated overnight at 4°C. The plate was incubated with WGA-biotin lectin (1:1000) (B-1025-5: Vector Labs) at room temperature for 1 hour, followed by the addition of diluted streptavidin-HRP and incubation for 45 minutes at room temperature. Within 30 minutes after addition of TMB (3,3',5,5'-tetramethylbenzidine) substrate, absorbance at 450 nm was measured using Flexstation 3 (Molecular Devices).

[0042] Lectin blotting: EVs were denatured in sample buffer containing 2-mercaptoethanol at 99°C for 5 minutes. The lysate was subjected to SDS-PAGE at 400 ng protein / lane. The separated proteins were transferred to a PVDF membrane and incubated with biotinylated lectin. After washing, the membrane was incubated with 1:5000 diluted streptavidin-HRP at room temperature for 20 minutes. The membrane was developed with Immobilon Western Chemiluminescent HRP Substrate (Merck) and observed with an imaging system (Amersham).

[0043] Mass spectrometry and proteome analysis. Proteome analysis was performed as previously reported. EV lysate was dissolved in an equal volume of M-PER buffer and suspended in SDS-PAGE sample buffer containing dithiothreitol (DTT). The sample was boiled at 99°C for 5 minutes and separated by SDS-PAGE. The gel was silver stained with Silver Stain KANTO III (Kanto Chemical). After slicing the gel, the sliced ​​gel was incubated at 37°C and digested with trypsin. The recovered peptides were desalted using Ziptip c18 (Millipore). The sample was analyzed using a nanoLC / MS / MS system (DiNa HPLC system, KYA TECH Corporation / QSTAR XL, Applied Biosystems). Protein identification was attempted using the mass data by Mascot search (MS / MS Ion Search).

[0044] Transmission Electron Microscopy (TEM) Formvar / carbon-coated copper grids (Cat. #6515, Nissin EM) were hydrophilized using a JFC-1600 Auto Fine Coater (JEOL). 3 μL of purified EV in PBS was placed on the hydrophilized grid and allowed to adsorb for 3 minutes. 500 μL of double-distilled H ２ The grid was washed with four successive drops of 0 and then negatively stained with four successive drops of 30 μL of 2.0% uranyl acetate. The 2.0% uranyl acetate was absorbed onto the grid with filter paper and then air-dried. The grid was imaged using a Tecnai G2 Spirit BioTWIN electron microscope (FEI) equipped with a Phurona CMOS camera (Emsis) operated at 120 kV. Raw data images were exported to TIFF format. Data analysis was performed using Fiji (ImageJ 1.53t).

[0045] Quantification and statistical analysis Statistical analysis and data visualization were performed using R software (v4.1.2). Fisher's exact test and Student's t-test were used to compare differences between two samples. Bartlett's test, one-way analysis of variance, and Tukey-Kramer test were used to examine differences in variance among the three groups. The pROC package (v1.18.4) in R software was used to estimate the diagnostic predictive value. The area under the ROC curve (AUC) was analyzed to determine the accuracy of the prediction model.

[0046] Example 1 Characteristics of Exosomes in the Blood of Patients with Depression The results of scanning electron microscopy observation of exosomes in the blood of healthy subjects (HS), patients with depression (acute phase: MDD-DP), and patients with depression (remission phase: MDD-REM) in the demographic data of Table 1 are shown in Figure 1. The average particle size (nm), protein concentration (μg / mL), and 10 exosomes in the blood of 10 healthy subjects, 10 patients with depression (acute phase), and 10 patients with depression (remission phase) were also measured. ９ The amount of protein (μg) per particle was investigated and the results are shown in FIG.

[0047] The results of Figures 1 and 2 show that there were no differences in exosome particle size or protein concentration among healthy individuals, patients with depression (acute phase), and patients with depression (remission phase).

[0048] [Example 2] Comparison of protein sugar chain patterns on exosomes Next, using multiple lectins (SBA, ConA, RCA-1, and WGA, which recognize N-type sugar chains, and UEA-I, PNA, and DBA, which recognize O-type sugar chains), the protein sugar chain patterns on exosomes derived from 10 healthy subjects and 10 patients with acute depression in the demographic data in Table 1 were examined by lectin blotting. Of these, the protein sugar chain patterns on exosomes derived from 5 healthy subjects and 5 patients with acute depression were examined by lectin blotting, and the results are shown in Figure 3.

[0049] As can be seen from FIG. 3, when ConA, RCA-1 and WGA, which are lectins that recognize N-glycans, were used, the band signals tended to be attenuated in patients with depression (acute phase) compared to healthy subjects.

[0050] Therefore, we focused on ConA, RCA-1, and WGA, which are lectins that recognize N-glycans in Figure 3, and focused on bands around 75 kDa or 250 kDa when using these lectins. The signal intensities of the bands in healthy subjects and patients with (acute) depression were examined, and the results are shown in Figure 4.

[0051] As shown in Figure 4, the signal intensity of the band in lectin WGA for proteins with a molecular weight of approximately 250 kDa was significantly reduced in patients with (acute) depression compared to healthy subjects. Therefore, glycosylated proteins on exosomes that are modified with glycosylation to which lectin WGA binds were identified as candidate biomarkers for depression.

[0052] Example 3 Comparison of Lectin WGA in the Acute and Remission Phases of Depression Based on the results of Example 2, analysis continued, focusing on lectin WGA. First, based on the demographic data in Table 1, lectin blotting was performed using exosomes derived from 10 healthy individuals, 10 patients with acute depression, and 10 patients with remission of depression. Lectin WGA was used as the lectin. The results of lectin blotting analysis of patients with depression in the remission and acute phases are shown in Figure 5. In Figure 5, the same numbers in the lane numbers indicate the same subject. The signal intensity of the band around 250 kDa was then measured, and the results of a comparison between the same patients with acute depression and those with remission of depression are shown on the left side of Figure 6, and the results of receiver operating characteristic (ROC) analysis based on the signal intensities of the bands of the same patients with acute depression and those with remission of depression are shown on the right side of Figure 6.

[0053] 5 and 6, it was confirmed that in the same patient, the number of glycans bound by lectin WGA in proteins present in exosomes increased when the patient transitioned from the acute phase to the remission phase. Furthermore, ROC analysis of patients with depression (acute phase) and patients with depression (remission phase) showed a high AUC value of 0.88.

[0054] Furthermore, the signal intensities of the bands around 250 kDa were measured and compared in 10 healthy subjects, 11 patients with depression (acute phase), and 10 patients with depression (remission phase). The results are shown on the left side of Figure 7, and the results of ROC analysis based on the signal intensities of the bands in healthy subjects and patients with depression (acute phase) are shown on the right side of Figure 7.

[0055] As shown in Figure 7, it was confirmed that when depression (acute phase) occurs, the sugar chains that bind to lectin WGA in proteins present in exosomes decrease, and when depression (remission phase) occurs, the above sugar chains increase. Furthermore, as shown in Figure 7, ROC analysis of healthy subjects and patients with depression (acute phase) showed a high AUC value of 0.88.

[0056] Furthermore, lectin blotting using lectin WGA was performed using exosomes derived from 20 healthy individuals and 21 patients with acute depression (MDD-DP) based on the demographic data in Table 2. The signal intensity of the band around 250 kDa in healthy individuals and patients with acute depression was measured and compared. The results are shown on the left in Figure 8, and the results of ROC analysis based on the signal intensity of the bands in healthy individuals and patients with acute depression are shown on the right in Figure 8.

[0057] As shown in Figure 8, the ROC analysis of healthy subjects and patients with depression (acute phase) based on lectin blotting using lectin WGA showed that the AUC value remained high at 0.87 even when the number of healthy subjects and the number of patients with depression (acute phase) increased by 10 each.

[0058] [Example 4] Identification of proteins on exosomes that bind to lectin WGA The above examples revealed that proteins on exosomes that bind to lectin WGA can be used to diagnose depression (acute phase) and to determine whether depression (acute phase) progresses to depression (remission phase). Therefore, we attempted to identify proteins on exosomes that bind to lectin WGA.

[0059] First, the above-mentioned "mass spectrometry and proteome analysis" was performed using exosomes derived from three healthy individuals based on the demographic data in Table 1. Figure 9 shows a photograph of the stained gel after separation by SDS-PAGE.

[0060] Next, the gel of the band around 250 kDa was sliced ​​and excised, and analyzed using a nanoLC / MS / MS system, and the protein was identified using Mascot search. The six identified glycoproteins are shown in Table 3.

[0061]

[0062] Of these, the ELISA analysis described above revealed that only von Willebrand factor (vWF) showed a difference in expression between healthy subjects and patients with acute depression. Figure 10 shows the results of ELISA analysis of vWF protein concentration on exosomes derived from five healthy subjects, 11 patients with acute depression (MDD-DP), and 10 patients with remission of depression (MDD-REM) from the demographic data in Table 1. Therefore, vWF was considered to be related to depression.

[0063] Furthermore, similarly to the above, the vWF protein concentrations on exosomes derived from 20 healthy individuals (HS) and 21 patients with acute depression (MDD-DP) were analyzed by ELISA based on the demographic data in Table 2. The results are shown on the left side of Figure 11, and the ROC analysis results based on the vWF protein concentrations on exosomes derived from the above healthy individuals and patients with acute depression (MDD-DP) are shown on the right side of Figure 11.

[0064] FIG. 11 confirms that healthy individuals can be distinguished from patients with depression (acute phase) by analyzing the vWF protein concentration on exosomes.

[0065] Example 5: Establishment of an objective method for evaluating the onset of depression and the acute and remission phases of depression using lectin WGA evaluation of vWF protein. Using lectin WGA, we first analyzed the concentrations of glycan-bound vWF protein on exosomes derived from 10 healthy subjects, 10 patients with acute depression, and 10 patients with remission of depression using the above-mentioned sandwich ELISA method based on the demographic data in Table 1. The results are shown in Figure 12. Furthermore, the results of ROC analysis of healthy subjects, patients with acute depression, and the same patients with remission and acute depression based on the glycan-bound vWF protein concentrations on exosomes are shown in Figure 13.

[0066] Furthermore, using lectin WGA, the concentrations of glycan-bound vWF protein on exosomes derived from 20 healthy subjects and 21 patients with acute depression (MDD-DP) were analyzed by the sandwich ELISA method described above based on the demographic data in Table 2. The results are shown on the left side of Figure 14. Furthermore, the results of ROC analysis of the 20 healthy subjects and 21 patients with acute depression (MDD-DP) based on the concentrations of glycan-bound vWF protein on exosomes are shown on the right side of Figure 14.

[0067] 12 to 14 reveal that a decrease in the abundance of N-glycans bound to vWF indicates that depression is in the acute phase, and that examining the recovery of the abundance of N-glycans bound to vWF makes it possible to diagnose whether depression has entered the remission phase. Therefore, it has become clear that measuring N-glycans bound to vWF present on extracellular vesicles in peripheral blood-derived plasma from patients with depression allows for more accurate diagnosis as objective data that eliminates the physician's experience and subjective factors, and also enables more accurate evaluation of treatment efficacy. In other words, it has become clear that vWF, particularly glycan-bound vWF, can be used as a biomarker for identifying depression.

[0068] Example 6: Discrimination of depression due to bipolar disorder In the above, judgments were made based on healthy subjects, acute depression, and depression in remission, but an attempt was made to distinguish between depression due to bipolar disorder.

[0069] Using the sandwich ELISA method described above, we analyzed the concentrations of glycan-bound vWF protein on exosomes derived from 10 healthy subjects (HS), 10 patients with acute depression (MDD-DP), and 10 patients with remission depression (MDD-REM) based on the demographic data in Table 1, as well as the concentrations of glycan-bound vWF protein on exosomes derived from 8 patients with bipolar disorder (BD). Bipolar disorder was defined as a group of bipolar disorders generally characterized by recurrent episodes of both manic phases (manic episodes) and depressive phases (depressive episodes) according to the international diagnostic criteria DSM-5-TR and ICD-11. The results are shown in Figure 15.

[0070] As shown in Figure 15, a decrease in the amount of N-glycans bound to vWF indicates that depression is in the acute phase, and it was confirmed that there is a significant difference between the depressive state of bipolar disorder and the acute phase of depression.

[0071] [Example 7] Discrimination of depression by measuring vWF in plasma In the above, it was revealed that depression can be diagnosed by analyzing the vWF protein concentration on exosomes. However, we investigated whether depression can also be diagnosed by analyzing the vWF protein concentration in plasma.

[0072] Between 9:00 AM and 12:00 PM, venous fasting blood samples were collected from 20 healthy volunteers and 21 patients with acute major depressive disorder (MDD-DP) based on the demographic data in Table 2. The blood samples were centrifuged at 2400 × g for 5 minutes to separate plasma from peripheral blood cells, yielding plasma. The vWF protein concentration in the plasma was then analyzed by the ELISA method described above. The results are shown in Figure 16.

[0073] 16 , it was revealed that it is difficult to distinguish depression from the vWF protein concentration in plasma. Therefore, it became clear that analyzing the vWF protein on exosomes is necessary to distinguish depression.

[0074] The present invention can be used for screening patients with depressive symptoms at their first non-psychiatric consultation, including with their primary care physician, to assist in the differential diagnosis of mood disorders, and to support early diagnosis and early intervention for depression.

Claims

1. A method for assisting in the diagnosis of depression, comprising the steps of measuring the amount of von Willebrand factor present in extracellular vesicles in a biological sample taken from a subject and comparing it with a predetermined value.

2. The method for assisting in the diagnosis of depression according to claim 1, wherein the von Willebrand factor is a glycosylated von Willebrand factor.

3. A method for assisting in the diagnosis of depression as described in claim 2, characterized in that the amount of glycan-bound von Willebrand factor is measured using a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the glycan.

4. The method for assisting in the diagnosis of depression according to claim 3, characterized in that the lectin is wheat germ agglutinin (WGA).

5. A method for assisting in the diagnosis of depression according to any one of claims 1 to 4, characterized in that the biological sample is plasma, serum or blood.

6. A method for assisting in the diagnosis of depression described in any one of claims 1 to 5, characterized in that a subject suffers from depression or has a history of being diagnosed with depression and determines whether or not the depression has gone into remission.

7. A kit for distinguishing depression, comprising an anti-von Willebrand factor antibody or a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the glycan of glycan-bound von Willebrand factor.

8. The kit for distinguishing depression according to claim 7, comprising an anti-von Willebrand factor antibody and a lectin that recognizes N-acetylglucosamine or sialic acid in the branched chain of the sugar chain of sugar-bound von Willebrand factor.

9. The kit for diagnosing depression according to claim 7 or 8, wherein the lectin is wheat germ agglutinin (WGA).

10. A kit for distinguishing depression according to any one of claims 7 to 9, wherein the anti-von Willebrand factor antibody is an anti-von Willebrand factor antibody not bound to a sugar chain.

11. Use of an anti-von Willebrand factor antibody or a lectin that recognizes the glycan of von Willebrand factor for diagnosing depression.